Calmodulin inhibitors increase the affinity of Merocyanine 540 for boar sperm membrane under non-capacitating conditions

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Calmodulin inhibitors increase the affinity of Merocyanine 540 for boar sperm membrane under non-capacitating conditions

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Title: Calmodulin inhibitors increase the affinity of Merocyanine 540 for boar sperm membrane under non-capacitating conditions
Author: González Fernández, Lauro; Macías García, Beatriz; Calle Guisado, Violeta; García Marín, Luis Jesús; Bragado González, María Julia
Abstract: El objetivo era comprobar si los inhibidores de la calmodulina (CaM), el calmidazolio (CZ) y la N-(6-aminohexil)-5-cloro-1-naftalenosulfonamida (W-7), pueden utilizarse para evaluar el trastorno de los lípidos mediante una citometría de flujo utilizando la merocianina 540 (M540). Los espermatozoides de jabalí se incubaron en condiciones no discapacitantes durante 10 min a temperatura ambiente con 1 μM CZ, 200 μM W-7, o 1 mM 8-bromoadenosina 3′,5′-monofosfato cíclico (8-Br-cAMP). Luego, los espermatozoides fueron 1) evaluados directamente, 2) centrifugados y lavados antes de la evaluación, o 3) diluidos con PBS antes de la evaluación. La evaluación directa mostró un aumento en la alta fluorescencia de M540 en los espermatozoides tratados con los inhibidores (4.7 ± 1.8 [control] vs. 70.4 ± 4.0 [CZ] y 71.4 ± 4.2 [W-7], % medio ± SD, P < 0.001); el lavado disminuyó el porcentaje de espermatozoides que mostraban alta fluorescencia de M540 para W-7 (4. 8 ± 2,2, % medio ± SD) pero no para CZ (69,4 ± 3,9, % medio ± SD, P < 0,001), y la dilución mostró un aumento de la alta fluorescencia M540 tanto para CZ como para W-7; el 8-Br-cAMP no indujo un aumento de los espermatozoides que mostraban alta fluorescencia M540. Por lo tanto, hay que tener especial cuidado cuando se utiliza M540 en espermatozoides con inhibidores de CaM.We aimed to test whether the calmodulin (CaM) inhibitors, calmidazolium (CZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), can be used to assess lipid disorder by flow cytometry using Merocyanine 540 (M540). Boar spermatozoa were incubated in non-capacitating conditions for 10 min at room temperature with 1 μM CZ, 200 μM W-7, or 1 mM 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP). Then, sperm were 1) directly evaluated, 2) centrifuged and washed prior to evaluation, or 3) diluted with PBS prior to evaluation. Direct evaluation showed an increase in high M540 fluorescence in spermatozoa treated with the inhibitors (4.7 ± 1.8 [control] vs. 70.4 ± 4.0 [CZ] and 71.4 ± 4.2 [W-7], mean % ± SD, P < 0.001); washing decreased the percentage of sperm showing high M540 fluorescence for W-7 (4.8 ± 2.2, mean % ± SD) but not for CZ (69.4 ± 3.9, mean % ± SD, P < 0.001), and dilution showed an increase in high M540 fluorescence for both CZ and W-7; 8-Br-cAMP did not induce a rise in sperm showing high M540 fluorescence. Therefore, special care must be taken when M540 is used in spermatozoa with CaM inhibitors.
URI: http://hdl.handle.net/10662/11465
Date: 2018


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